Analysis of binding domain and function of chimeric mu/kappa opioid receptors to ohmefentanyl stereoisomers

To investigate specific domains in mu opioid receptors that accounted for selective binding of three stereoisomers of ohmefentanyl (Ohm9204, Ohm9202, and Ohm9203) and study the function of chimera II.

Rat mu and kappa opioid receptors (RMOR, RKOR) and four mu/kappa chimeric receptors (chimeras) I, II, III, and IV were transiently expressed in COS-1 cells. The binding ability and binding domain of receptor to ligands were determined by radioactive ligand and receptor binding experiments. Through measuring cellular cAMP levels, we studied the function of chimera II in mediating signal transduction.

Binding affinities of four chimeric receptors were similar to wild type opioid receptors (RMOR and RKOR). The binding affinities of Ohm9204 and Ohm9202 to chimera II were similar to that of RMOR. The binding affinities of Ohm9203 to all six receptors were low. U50488 possessed high binding affinity to chimera I, however dynorphie A(1-9) had some binding affinity to chimera II that was similar to RKOR, which indicated the domains of RKOR accounting for selectively binding to peptide ligand dynorphie A(1-9) and nonpeptide ligand U50488 were different. The efficacy of Ohm9204 and Ohm9203 on inhibiting forskolin-stimulated cAMP accumulation in cells transfected with chimera II was similar to that in cells transfected with RMOR.

Replacing 194-268 residues of RMOR with 185-262 residues of RKOR does not influence the ability of mu opioid receptor to bind Ohm9204 and Ohm9202 and the receptor mediated inhibition of cellular cAMP level.